Rationale: Gastric cancer is a leading cause of cancer death and ranks second in the overall cancer mortality worldwide. The GI (gastrointestinal) peptide hormone gastrin (both processed and unprocessed forms) is regarded as an important contributing factor towards the patho-physiology of GI malignancies. Studies with mice over-expressing the mature amidated form of gastrin were shown to produce invasive gastric carcinoma, which synergized with Helicobacter infection to promote carcinoma progression. These effects of gastrin pose a major clinical concern, since an increase in serum gastrin levels is a common side effect of the use of proton pump inhibitors (PPIs) or Helicobacter pylori infection. Thus the signaling pathways by which gastrin mediates cancer progression, though still poorly understood, represent an important target for the development of drugs to combat gastric cancer. The major focus of this proposal is to elucidate the signaling mechanism by which gastrin promotes tumor progression and metastasis in gastric cancer cells, and to target this signaling axis in mouse models of gastric cancer. Our recent studies indicated that: (i) treatment with amidated gastrin (G17) potently induces gastric cancer cell migration and invasion; (ii) G17-induced migration is associated with activation of all 3 MAPK pathways including ERK, p38 and JNK; (iii) G17 stimulation results in activation of the MAP3K member MLK3, which is an upstream activator of JNK, (iv) G17-induced migration operates via MLK3/JNK1 axis in vitro; (v) G17 activates the JNK downstream c-Jun via releasing some factor and (vi) conditioned media obtained from G17-treated gastric cancer cells showed increased IL-10 release by G17. Hypothesis: Based on these preliminary data we hypothesize that G17-induced gastric cancer cell growth (proliferation), migration and invasion operates through activation of MLK3/JNK1 signaling. Specific Objectives: To elucidate the signaling mechanism by which G17 induces gastric carcinoma progression, we propose the following 3 specific aims: (1) to determine whether G17-induced migration, invasion, EMT and growth involve the MLK3/JNK1 axis in vitro; (2) to elucidate the mechanism by which G17 activates MLK3/JNK1 axis and the role of cytokines in mediating this; (3) to determine the effect of hypergastrinemia on the progression of gastric cancer in vivo and the effect of inhibition of MLK3/JNK1 pathways, either by pharmacological inhibitors or via gene silencing on this progression. Procedures to be used: The proposed studies will be performed using a broad range of technical approaches, including migration, invasion and cell proliferation assays, Western analyses, DNA transfection, luciferase and kinase assays, immunoprecipitation, SiRNA technology, subcutaneous xenografts and tail vein injections. Significance of potential new findings: The fact that hypergastrinemia promotes tumor progression poses a major clinical concern in the setting of increased PPI use and H.pylori infection. No information is currently available regarding the involvement of MLK3/JNK axis in promoting gastric cancer growth and progression. The successful completion of these studies will determine whether this axis promotes gastrin-induced gastric carcinoma progression. The results from these studies can further promote the detailed investigation of the potential use of available MLK/JNK pathway inhibitors as safe, noninvasive drugs for the treatment of patients with gastric as well as other gastrin- dependent GI malignancies. PUBLIC HEALTH RELEVANCE: The proposed studies are of significant importance to the VA patient population, since GI cancer is prevalent among aging veterans. Surgical resection is the only form of therapy currently available to treat gastric or GI cancers, which is effective only if the tumor is detected early. The long-term focus of our studies is to develop a noninvasive form of curative therapy for treating VA patients with gastric or other GI malignancies. To achieve this goal, it is important to gain a better understanding of the molecular events that lead to the development and progression of these diseases. Since the GI peptide hormone gastrin is known to promote progression of gastric cancers, the studies described here will be focused on understanding the detailed mechanism by which gastrin mediates this function. These studies will be performed utilizing both cell culture (in vitro) and animal models of gastric cancer (in vivo). Once these mediators are identified, drugs that can target these mediators can be designed in the future, and utilized as safer, noninvasive therapeutic drugs for treating gastric cancer.